Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

• US 20150119258A1
• Filed: 07/08/2014
• Published: 04/30/2015
• Est. Priority Date: 12/15/2009
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fodor, Stephen P.A., Fu, Glenn K.
• Application Number:
  US14/326,452
• Publication Number:
  US 20150119258A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

Claims

19. A method of sequencing, comprising:

a) appending a degenerate base region (DBR) to both ends of a plurality of target nucleic acid molecules in a genomic sample, wherein said DBR comprises a sequence comprising at least one nucleotide base selected from;

R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein said appending produces a population of asymmetrically tagged nucleic acid molecules, wherein each of the asymmetrically tagged nucleic acid molecules comprises;

(i) a target polynucleotide,

(ii) a first DBR sequence at the 5′

end of the target polynucleotide and

(iii) a second DBR sequence at the 3′

end of the target polynucleotide, wherein;

in each of said asymmetrically tagged nucleic acid molecules the first DBR sequence is different from the second DBR sequence; and

the different asymmetrically tagged nucleic acid molecules in said population have different first DBR sequences and different second DBR sequences, relative to one another;

b) amplifying at least some of said asymmetrically tagged nucleic acid molecules, thereby producing a population of amplified asymmetrically tagged nucleic acid molecules; and

c) sequencing a plurality of the amplified asymmetrically tagged nucleic acid molecules to produce a plurality of sequences, wherein the sequencing step provides, for each of the asymmetrically tagged nucleic acid molecules that are sequenced;

(i) the nucleotide sequence of at least a portion of a target polynucleotide;

(ii) the nucleotide sequence of the first DBR sequence; and

(iii) the nucleotide sequence of the second DBR sequence.

1. A method of sequencing, comprising:

a) attaching a label-tag to both ends of a plurality of target nucleic acid molecules in a genomic sample,

wherein the label-tag comprises natural nucleotide bases and/or chemically or biochemically modified, non-natural, or derivatized nucleotide bases, and

wherein the attaching produces a population of tagged nucleic acid molecules, wherein each of the tagged nucleic acid molecules comprises;

(i) a target polynucleotide;

(ii) a first label-tag at the 5′

end of the target polynucleotide; and

(iii) a second label-tag at the 3′

end of the target polynucleotide,

wherein;

in each of the tagged nucleic acid molecules the first label-tag is different from the second label-tag; and

the different tagged nucleic acid molecules in the population have different first label-tags and different second label-tags, relative to one another;

b) amplifying at least some of the tagged nucleic acid molecules, thereby producing a population of amplified tagged nucleic acid molecules; and

c) detecting the plurality of amplified labeled-targets by sequencing, thereby producing a plurality of readouts, wherein the plurality of readouts comprises;

(i) at least a portion of the target;

(ii) the first label-tag; and

(iii) the second label-tag.

2. The method of claim 1, wherein the attaching step is done by ligating a set of adaptors that comprise the label-tag to the plurality of target nucleic acid molecules.

3. The method of claim 1, wherein the attaching is done by extending a set of primers that comprises the label-tags, using an initial nucleic acid sample as a template.

4. The method of claim 3, wherein the initial nucleic acid sample is an amplification product.

5. The method of claim 1, wherein the method comprises, prior to the attaching step (a), enriching for the target nucleic acid molecules from an initial nucleic acid sample.

6. The method of claim 5, wherein the initial nucleic acid sample is an amplification product.

7. The method of claim 1, wherein the label-tags comprise at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases is selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases.

8. The method of claim 7, wherein the label-tags comprise from 2 to 20 nucleotide bases, wherein each of the 2 to 20 nucleotide bases is selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases.

9. The method of claim 1, wherein the tagged nucleic acid molecules further comprise a unique sequence tag that is used to distinguish target DNA molecules from different samples.

10. The method of claim 9, wherein the tagged genomic sample is a mixed sample comprising nucleic acid molecules from different samples, wherein each of the samples is associated with a different unique sequence tag.

11. The method of claim 9, wherein each of the different samples is derived from a human subject.

12. The method of claim 9, wherein each of the different samples comprises polynucleotides from tumor cells.

13. The method of claim 1, wherein the genomic sample comprises polynucleotides from a tumor cell.

14. The method of claim 1, wherein the genomic sample comprises polynucleotides from bacteria and/or polynucleotides encoding viral epitopes.

15. The method of claim 1, wherein the genomic sample comprises human genomic DNA.

16. The method of claim 1, wherein detecting the plurality of amplified labeled-targets by sequencing comprises sequencing a plurality of the amplified tagged nucleic acid molecules on a next-generation sequencing platform.

17. The method of claim 1, wherein the amplifying step (b) is done by polymerase chain reaction.

18. The method of claim 1, wherein the label-tag is used to correct estimation errors.

20. The method of claim 19, wherein the appending step is done by ligating a set of adaptors that comprise said DBR to said plurality of target nucleic acid molecules.

21. The method of claim 19, wherein said appending is done by extending a set of primers that comprises said DBR, using an initial nucleic acid sample as a template.

22. The method of claim 21, wherein said initial nucleic acid sample is an amplification product.

23. The method of claim 19, wherein the method comprises, prior to the appending step (a), enriching for said target nucleic acid molecules from an initial nucleic acid sample.

24. The method of claim 23, wherein said initial nucleic acid sample is an amplification product.

25. The method of claim 19, wherein said DBR comprises at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases are selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

26. The method of claim 25, wherein said DBR comprises from 3 to 10 nucleotide bases, wherein each of the 3 to 10 nucleotide bases is selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

27. The method of claim 19, wherein said asymmetrically tagged nucleic acid molecules further comprise a unique multiplex identifier (MID) sequence that identifies the source of a nucleic acid molecule to which it is appended.

28. The method of claim 27, wherein said genomic sample is a pooled sample comprising nucleic acid molecules from several different sources, where each of said sources is associated with a different MID sequence.

29. The method of claim 27, wherein each of the sources is derived from a human subject.

30. The method of claim 27, wherein each of the sources is derived from different sections of a tumor.

31. The method of claim 27, wherein each of the sources is derived from different tumors of a subject.

32. The method of claim 27, wherein each of the sources is derived from a subject at different times.

33. The method of claim 19, wherein the genomic sample comprises polynucleotides from a tumor.

35. The method of claim 19, wherein the genomic sample comprises polynucleotides from a microorganism and/or a virus.

36. The method of claim 19, wherein the sequencing step c) comprises sequencing a plurality of the amplified asymmetrically tagged nucleic acid molecules on a next-generation sequencing platform.

37. The method of claim 19, wherein the amplifying step (b) is done by polymerase chain reaction.

38. The method of claim 19, wherein the DBR comprises an error-correcting code.

35-1. The method of claim 19, wherein the genomic sample comprises human genomic DNA.